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Examining the Differential DNA Yield Rates Between Human Skeletal Elements Over Increasing Post Mortem Intervals

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The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Developing an Empirically Based Ranking Order for Bone Sampling: Examining the Differential DNA Yield Rates Between Human Skeletal Elements Over Increasing Post Mortem Intervals Author(s): Amy Z. Mundorff, Ph.D., Jonathan Davoren, M.S., Shannon Weitz, B.S. Document No.: 241868 Date Received: April 2013 Award Number: 2010-DN-BX-K229 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this Federally- funded grant report available electronically. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Final Technical Report Document Title: Developing an Empirically Based Ranking Order for Bone Sampling: Examining the Differential DNA Yield Rates Between Human Skeletal Elements Over Increasing Post Mortem Intervals Authors: Amy Z. Mundorff, Ph.D., Jonathan Davoren, M.S., Shannon Weitz, B.S. Award Number: 2010-DN-BX-K229 NIJ Award 2010-DN-BX-K229 Final Technical Report 1 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Abstract This study established an empirically based ranking of skeletal elements according to each bone’s capacity to provide usable genetic material for a DNA identification and assessed how these results varied over longer post mortem intervals (PMI). Currently, selecting skeletal elements for DNA testing is based on the collective wisdom of practitioners who suggest weight-bearing long bones over all other elements. Recent evidence questions the accuracy of this collective wisdom. This study evaluated the differential yield and preservation of DNA by skeletal element in a single individual, replacing intuition with empirically based data to increase the likelihood of successfully generating a DNA profile from skeletal material. A two- phase study was conducted to accomplish these goals. Phase 1 entailed analyzing the DNA yield rates of different skeletal elements, from 3 recently skeletonized individuals, to generate an overall rank order from most successful to least successful. Phase 2 was designed to determined if the same elements from Phase 1 were equally successful at longer post mortem intervals (0-3, 4-10, 11-20, and 20+ years) and to give an indication how DNA degradation occurs over time. Using a single sampling (0.2 g) and a single amplification targeting 2.0 ng, both the quantity and quality of DNA were analyzed to determine which bones most successfully yielded full profiles (15 loci + amelogenin). Phase 1 results indicated that the quantity and quality of DNA obtained from different skeletal elements is highly variable. Small, predominantly cancellous bones such as a phalanx, patella, and tarsal’s, out performed the femur, tibia, and other long bones, which are comprised mostly of the ‘preferred’ dense cortical bone. The bones that ranked at the top (full profiles) consistently for all three individuals were considered for Phase 2. Only ten NIJ Award 2010-DN-BX-K229 Final Technical Report 2 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. bones per individual would be tested in Phase 2, however, 23 elements and 4 teeth qualified by achieving the ‘most successful’ rank in Phase 1. Therefore elements were selected by yield per mass of sample, average RFU value for each element, and skeletal representation. A cervical vertebra, middle rib, patella, 4th metacarpal, 1st distal hand phalanx, 4th metatarsal, talus, 1st cuneiform, tibia, and femur were selected for Phase 2. Phase 1 results for the tibia and femur did not reach the specifications to qualify for Phase 2 however, they are elements practitioners typically prefer to sample and therefore were included for comparison. Three skeletons from each of the four post mortem intervals (12 total) were selected. Only skeletons with similar demographics (age, sex, etc.) to the initial three individuals from Phase 1 were considered for Phase 2. Phase 2 results show that most elements produced less complete DNA profiles as the PMI increased. However, the elements that produced the best results in Phase 1 continued to perform well in later post mortem intervals, retaining their positional rank in relation to each other. This suggests that DNA degrades consistently over time. Interestingly, the distal hand phalanx and the 4th metatarsal, bones typically not sampled, were the only elements to produce full profiles in the final PMI of 20+ years. This research project determined which skeletal elements, within a single individual, were most likely to provide both the quantity and quality of DNA needed to produce DNA profiles from increasing post mortem intervals. The results provide much needed guidance on which elements are best suited for DNA testing. Cancellous bones, typically dismissed as a potential DNA source in favor of dense cortical bones, should be reconsidered when sampling. By determining which skeletal element is most NIJ Award 2010-DN-BX-K229 Final Technical Report 3 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. likely to yield an STR profile allowing identification, expensive and time-consuming retesting can be avoided. NIJ Award 2010-DN-BX-K229 Final Technical Report 4 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Table of Contents ABSTRACT................................................................................................................... 2 TABLE OF CONTENTS................................................................................................ 5 EXECUTIVE SUMMARY .............................................................................................. 6 IMPLICATIONS FOR POLICY AND PRACTICE .................................................... 16 I. INTRODUCTION............................................................................................... 18 II. MATERIALS AND METHODS......................................................................... 30 III. RESULTS........................................................................................................ 42 IV. CONCLUSIONS. ............................................................................................ 62 V. REFERENCES ................................................................................................ 66 VI. DISSEMINATION OF RESEARCH FINDINGS.............................................. 77 NIJ Award 2010-DN-BX-K229 Final Technical Report 5 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. EXECUTIVE SUMMARY Description of the problem Selecting the best bone to sample in order to maximize the chances of acquiring sufficient DNA for an identification can be challenging. Recent advances in genetic sequencing techniques have been driven by large-scale identification efforts focused primarily on nuclear DNA from osseous material (Whitaker et al. 1995; Hsu et al. 1999; Hoff-Olsen et al. 2003; Piccinini et al. 2004; Davoren et al. 2007). Few laboratories can conduct this type of analysis and for many jurisdictions these tests remain prohibitively expensive. Yet, as awareness of DNA capabilities continues to grow with investigators and in the popular culture, the number of unidentified remains requiring DNA identification is also growing. However, bones degrade over time, which increases the difficulty of securing an identification through DNA. Previous research has established a practice of preferentially sampling dense, cortical, weight-bearing long bones, such as the femur. While this research has been important, it has focused only on selected elements of the skeleton. New evidence indicates that the practice of preferentially sampling cortical bone may be suboptimal. A retrospective study examining a subset of the World Trade Center remains indicated that bones such as the phalanx and the patella yield DNA as well or better than dense cortical bone such as the femur (Mundorff et al. 2009).
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